RESUMEN
Scutellarin is used to treat brain ischaemia. However, its underlying mechanism of action remains unclear. This study aimed to elucidate the potential mechanism of action of scutellarin in brain ischaemia through network pharmacology and experimental verification. The JAK2/STAT3 signalling pathway was identified and experimentally verified. Expression of JAK2/STAT3 signalling related proteins in TNC-1 astrocytes with BV-2 microglia-conditioned medium (CM), CM + lipopolysaccharide (LPS) (CM + L), and CM pretreated with scutellarin + LPS (CM + SL) was analysed by Western Blot and immunofluorescence staining. Expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 were evaluated in astrocytes pre-treated with AG490. Middle cerebral artery occlusion (MCAO) in rats was performed in different experimental groups to detect expression of the above biomarkers. Network pharmacology suggested that the JAK2/STAT3 signalling pathway is one of the mechanisms by which scutellarin mitigates cerebral ischaemic damage. In TNC-1 astrocytes, p-JAK2 and p-STAT3 expression were significantly up-regulated in the CM + L group. Scutellarin promoted the up-regulation of various markers and AG490 neutralised the effect of scutellarin. In vivo, up-regulation of p-JAK2 and p-STAT3 after ischaemia is known. These results are consistent with previous reports. Scutellarin further enhanced this upregulation at 1, 3, and 7 d after MCAO. Scutellarin exerts its therapeutic effects on cerebral ischaemia by activating the astrocyte JAK2/STAT3 signalling, which provides a firm experimental basis for its clinical application.
Asunto(s)
Lesiones Encefálicas , Isquemia Encefálica , Animales , Ratas , Farmacología en Red , Lipopolisacáridos , Isquemia Encefálica/tratamiento farmacológico , Medios de Cultivo Condicionados , Janus Quinasa 2RESUMEN
Scutellarin, an herbal agent, is known to possess anti-oxidant and anti-inflammatory properties. In activated microglia, it has been reported that this is achieved through acting on the MAPKs, a key pathway that regulates microglia activation. This study sought to determine if scutellarin would affect the commonly described microglia phenotypes, namely, M1 and M2, thought to contribute to pro- and anti-inflammatory roles, respectively. This is in consideration of its potential effect on the polarization of microglia phenotypes that are featured prominently in cerebral ischemia. For this purpose, we have used an experimentally induced cerebral ischemia rat model and LPS-stimulated BV-2 cell model. Thus, by Western blot and immunofluorescence, we show here a noticeable increase in expression of M2 microglia markers, namely, CD206, Arg1, YM1/2, IL-4 and IL-10 in activated microglia both in vivo and in vitro. Besides, we have confirmed that Scutellarin upregulated expression of Arg1, IL-10 and IL-4 in medium supernatants of BV-2 microglia. Remarkably, scutellarin treatment markedly augmented the increased expression of the respective markers in activated microglia. It is therefore suggested scutellarin can exert the polarization of activated microglia from M1 to M2 phenotype. Because M1 microglia are commonly known to be proinflammatory, while M2 microglia are anti-inflammatory and neuroprotective effect, it stands to reason therefore that with the increase of M2 microglia which became predominant by scutellarin, the local inflammatory response is ameliorated. More importantly, we have found that scutellarin promotes the M2 polarization through inhibiting the JNK and p38 signaling pathways, and concomitantly augmenting the ERK1/2 signaling pathway. This lends its strong support from observations in LPS activated BV-2 microglia treated with p38 and JNK inhibitors in which expression of M2 markers was increased; on the other hand, in cells subjected to ERK1/2 inhibitor treatment, the expression was suppressed. In light of the above, MAPKs pathway is deemed to be a potential therapeutic target of scutellarin in mitigating microglia mediated neuroinflammation in activated microglia.
Asunto(s)
Isquemia Encefálica , Microglía , Ratas , Animales , Microglía/metabolismo , Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Interleucina-4 , Antiinflamatorios/farmacología , Isquemia Encefálica/metabolismoRESUMEN
BACKGROUND: Gastrodin (GAS) has been proven to play a therapeutic role in a variety of neurological diseases by affecting activated astrocytes, however, the underlying mechanisms have not been fully illustrated. This study aimed to investigate if GAS exerts the neuroprotective effect through regulating the Notch signaling pathway involved in reactive astrocytes. METHODS: Astrocyte cell lines (TNC1 cells) were cultured in vitro. The hypoxic-ischemic cell model was prepared using the oxygen-glucose deprivation (OGD) method, GAS's pretreatment concentration was 0.34 mM, intervention for 1 hour. Cell counting kit-8 (CCK-8) assay, Transwell migration assay, immunofluorescent staining (double staining), and Western blotting were used to observe the effects of OGD or GAS interference on the function of astrocytes, and the changes of key protein expressions in the Notch signaling pathway were analyzed. RESULTS: GAS had no obvious toxic effect on TNC1 astrocytes under physiological conditions. Following OGD, GAS can not only improve cell viability and migration, but also regulate the production of inflammatory mediators. We also found that OGD significantly increased the expression of key proteins related to the Notch signaling pathway, Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK), transcription factor hairy and enhancer of split-1 (Hes-1) in TNC1 astrocytes, which was significantly inhibited by GAS. In addition, GAS inhibited the OGD-induced expression of TNC1 astrocyte tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß), and enhanced the expression of nutrient factors, including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1). The Notch signaling pathway specific inhibitor, N-[N-(3,5-Difluorophenacetyl)-1-alany1]-S-phenyglycine t-butylester (DAPT), could significantly enhance the effect of GAS on TNC1 astrocytes after OGD, such as the inhibition of inflammatory factors and the up-regulation of neurotrophic factors. CONCLUSIONS: GAS exerts dual effects on astrocytes via regulation of the Notch signaling pathway. We found that it could inhibit the pro-inflammatory factors mediated by astrocytes, and also promote the secretion of neurotrophic factors by astrocytes. These results provide a new biological mechanism for the treatment of neuroinflammatory diseases by GAS.
RESUMEN
In response to hypoxic-ischemic brain damage (HIBD), microglia activation and its mediated inflammation contribute to neuronal damage. Inhibition of over-activated microglia is deemed to be a potential therapeutic strategy. Our previous studies showed that gastrodin efficiently depressed the neuroinflammation mediated by activated microglia in HIBD neonatal rats. The underlying mechanisms through which gastrodin acts on activated microglia have not been fully elucidated. This study is designed to determine whether gastrodin would regulate the Notch signaling pathway and Sirtuin3 (Sirt3), which are implicated in regulating microglia activation. The present results showed that gastrodin markedly suppressed the expression of members of Notch signaling pathway (Notch-1, NICD, RBP-JK and Hes-1) in activated microglia both in vivo and in vitro. Conversely, Sirt3 expression was enhanced. In BV-2 microglia treated with a γ-secretase inhibitor of Notch pathway- DAPT, the expression of RBP-JK, Hes-1, and NICD was suppressed in activated microglia. Treatment with DAPT and gastrodin further decreased NICD and Hes-1 expression. Sirt3 expression was also decreased after DAPT treatment. However, Sirt3 expression in activated BV-2 microglia given a combined DAPT and gastrodin treatment was not further increased. In addition, combination of DAPT and Gastrodin cumulatively decreased tumor necrosis factor-α (TNF-α) expression. The results suggest that gastrodin regulates microglia activation via the Notch signaling pathway and Sirt3. More importantly, interference of the Notch signaling pathway inhibited Sirt3 expression, indicating that Sirt3 is a downstream gene of the Notch signaling pathway. It is suggested that Notch and Sirt3 synergistically regulate microglia activation such as in TNF-α production.
Asunto(s)
Alcoholes Bencílicos/farmacología , Glucósidos/farmacología , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Microglía/efectos de los fármacos , Receptor Notch1/fisiología , Transducción de Señal/efectos de los fármacos , Sirtuinas/fisiología , Animales , Animales Recién Nacidos , Alcoholes Bencílicos/farmacocinética , Arteria Carótida Común , Células Cultivadas , Corteza Cerebral/patología , Cuerpo Calloso/patología , Diaminas/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Glucósidos/farmacocinética , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/patología , Ligadura , Lipopolisacáridos/farmacología , Microglía/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Receptor Notch1/biosíntesis , Receptor Notch1/genética , Sirtuinas/biosíntesis , Sirtuinas/genética , Tiazoles/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Scutellarin, an herbal compound, can effectively suppress the inflammatory response in activated microglia/brain macrophage(AM/BM) in experimentally induced cerebral ischemia; however, the underlying mechanism for this has not been fully clarified. We sought to elucidate if scutellarin would exert its anti-inflammatory effects on AM/BM through the MAPKs pathway. MATERIALS AND METHODS: Western blot and immunofluorescence labeling were used to determine the expression of the MAPKs pathway in AM/BM in rats subjected to middle cerebral artery occlusion (MCAO) also in lipopolysaccharide (LPS)-activated BV-2 microglia in vitro. Furthermore, expression of p-p38 along with that of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta(IL-1ß), and inducible nitric oxide synthase (iNOS) in LPS-activated microglia subjected to pretreatment with p38 inhibitor SB203580, p38 activator sc-201214, scutellarin, or a combination of them was evaluated. FINDINGS: Scutellarin markedly attenuated the expression of p-p38, p-JNK in AM/BM in MCAO rats and in vitro. Conversely, p-ERK1/2 expression level was significantly increased by scutellarin. Meanwhile, scutellarin suppressed the expression of proinflammatory mediators including iNOS, TNF-α, and IL-1ß in AM/BM. More importantly, SB203580 suppressed p-p38 protein expression level in LPS-activated BV-2 microglia that was coupled with decreased expression of proinflammatory mediators (TNF-α, iNOS) in LPS-activated BV-2 microglia. However, p38 activator sc-201214 increased expression of proinflammatory mediators TNF-α, iNOS, and IL-1ß. Interestingly, the decreased expression of both proinflammatory markers by p38 MAPK inhibitor and increased expression of proinflammatory markers by p38 MAPK activator were compatible with that in BV-2-activated microglia pretreated with scutellarin. CONCLUSIONS: The results suggest that scutellarin down-regulates the expression of proinflammatory mediators in AM/BM through suppressing the p-JNK and p-p38 MAPKs. Of note, the anti-inflammatory effect of p38 MAPK inhibitor and scutellarin is comparable. Besides, p38 MAPKs activator reverses the effect of scutellarin. Additionally, scutellarin increases p-ERK1/2 expression that may be neuroprotective.